Molecular double-check strategy for the identification and characterization of European Lyssaviruses

Melina Fischer, Conrad M. Freuling, Thomas Müller, Anne Wegelt, Engbert A. Kooi, Thomas Bruun Rasmussen, Katja Voller, Denise A. Marston, Anthony R. Fooks, Martin Beer, Bernd Hoffmann

    Research output: Contribution to journalJournal articleResearchpeer-review

    280 Downloads (Pure)

    Abstract

    The “gold standard” for post-mortem rabies diagnosis is the direct fluorescent antibody test (FAT). However, in the case of ante-mortem non-neural sample material or decomposed tissues, the FAT reaches its limit, and the use of molecular techniques can be advantageous. In this study, we developed and validated a reverse transcription PCR cascade protocol feasible for the classification of samples, even those for which there is no epidemiological background knowledge. This study emphasises on the most relevant European lyssaviruses.

    In a first step, two independent N- and L-gene based pan-lyssavirus intercalating dye assays are performed in a double-check application to increase the method's diagnostic safety. For the second step, characterization of the lyssavirus positive samples via two independent multiplex PCR-systems was performed. Both assays were probe-based, species-specific multiplex PCR-systems for Rabies virus, European bat lyssavirus type 1 and 2 as well as Bokeloh bat lyssavirus. All assays were validated successfully with a comprehensive panel of lyssavirus positive samples, as well as negative material from various host species.

    This double-check strategy allows for both safe and sensitive screening, detection and characterization of all lyssavirus species of humans and animals, as well as the rapid identification of currently unknown lyssaviruses in bats in Europe.
    Original languageEnglish
    JournalJournal of Virological Methods
    Volume203
    Pages (from-to)23-32
    ISSN0166-0934
    DOIs
    Publication statusPublished - 2014

    Cite this

    Fischer, M., Freuling, C. M., Müller, T., Wegelt, A., Kooi, E. A., Rasmussen, T. B., Voller, K., Marston, D. A., Fooks, A. R., Beer, M., & Hoffmann, B. (2014). Molecular double-check strategy for the identification and characterization of European Lyssaviruses. Journal of Virological Methods, 203, 23-32. https://doi.org/10.1016/j.jviromet.2014.03.014